Plasminogen activator inhibitor type-1 (PAI-1) is the major physiologic regulator of the plasmin-based pericellular proteolytic cascade and a critical element in pathologic angiogenesis in vivo. PAI-1 controls stromal proteolysis (i.e., maintains an angiogenic "scaffold") and stabilizes nascent capillary vessel structure. Our findings as well as others indicate that PAI-1 negatively modulates a plasmin/matrix metalloproteinase (MMP) cascade that regulates collagen degradation, capillary regression and endothelial apoptosis. Our continued definition of mechanistic controls on endothelial PAI-1 gene expression may lead to new therapies for the treatment of diseases in which exuberant or non-resolving angiogenesis is a major pathogenic feature (e.g., arthritis, psoriasis, cancer, cardiovascular and pulmonary fibrosis, diabetic retinopathy, macular degeneration, excessive scaring disorders). In the previous funding period, we established that TGF-p1- stimulated PAI-1 expression requires cooperative EGFR/pp60G"s/c and MEK signaling and involved the rho kinase pp160ROCK. PAI-1 transcription in response to TGF-p1, moreover, required binding of upstream stimulatory factor (USF) to a critical E box motif (CACGTG) in the PAI-1 promoter. Chromatin immunopre- cipitation confirmed that PAI-1 is a direct USF target gene and that TGF-pl-induced PAI-1 expression involves a subtype switch (USF1->USF2) at the E box site. USF, furthermore, formed transient complexes with pERK and interference with EGFR, ppSO0"5"1 or p160ROCK activity or ERK/p38 signaling ablated induced PAI-1 expression. Importantly, targeted PAI-1 down-regulation with siRNA or antisense constructs or expression of dominant-negative USF mutants as well as use of PAI-1 neutralizing reagents accelerated plasminogen-induced collagen degradation and vessel regression. Our hypothesis is that TGF-B1 stimulates PAI-1 transcription through cooperative pathways that modulate the activation state of the EGFR and regulate MAP kinase/USF interactions. The following aims will address this hypothesis: (1) to characterize MAP kinase/USF interactions and phosphorylation site requirements in endothelial cells that regulate USF/DNA binding and PAI-1 transcription, (2) to determine the mechanism by which TGF-R.1 activates the EGFR and r/?o/p160ROCK signaling. Our continued clarification of mechanistic controls on PAI-1 gene expression may lead to new targeted therapies for diseases in which excessive or non-resolving angiogenesis is a major pathogenic feature.